The invention is directed to a process for the bioconversion of a carbon source, which is preferably a carbohydrate or another carbon containing compound into a target organic compound by an enzymatic process, preferably in the absence of productive living cells. The target organic compound is preferably a hydrophobic, a hydrophilic or an intermediate chemical compound.
Hydrophobic chemicals according to the invention comprise, without limitation, C4 alcohols such as n-butanol, 2-butanol, and isobutanol, and other chemicals that have a limited miscibility with water. Limited miscibility means that at room temperature not more than 20% (w/w) can be mixed with water without phase separation. Hydrophilic and intermediate chemicals according to the invention comprise, without limitation ethanol and other chemicals.
n-Butanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 7-8% at RT) in water. n-Butanol is used as an intermediate in the production of chemicals, as a solvent and as an ingredient in formulated products such as cosmetics. n-Butanol is used in the synthesis of acrylate/methacrylate esters, glycol ethers, n-butyl acetate, amino resins and n-butylamines. n-Butanol can also be used as a fuel in combustion engines due to low vapor pressure, high energy content and the possibility to be blended with gasoline at high concentrations.
2-Butanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 12% at RT) in water. 2-Butanol is used as solvent for paints and coatings as well as food ingredients or in the production of 1-butene.
Isobutanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 9-10% at RT) in water. Isobutanol is used as solvent or as plasticizer. It is also used in the production of isobutene which is a precursor for the production of MTBE or ETBE.
n-Butanol can be produced using solventogenic Clostridia, such as C. acetobuylicum or C. beijerinckii, typically producing a mixture of n-butanol, acetone and ethanol. Butanol production using solventogenic clostridia has several drawbacks: (i) Product isolation from dilute aqueous solution is very expensive as it is either elaborate (e.g. using membrane processes) or energy consuming (e.g. using distillation). (ii) The yield is low as significant parts of the substrate go into the formation of by-products such as acetone, ethanol, hydrogen and biomass. (iii) The productivity of butanol production is low due to limited cell titres. (iv) The complex metabolism limits metabolic engineering for higher productivity and yield. (v) Limited process stability often leads to production losses and sterility is difficult to maintain. (vi) The biphasic nature of clostridial growth limits process flexibility and productivity.
Several approaches exist to overcome the limitations of classical fermentative butanol production. For example, WO2008/052596 describes recombinant modification of Clostridia for improved yield. Selection or engineering of variants for higher Butanol resistance is, for example, described in WO 2008/006038.
The cell-free production of chemicals has been shown as early as 1897 when Eduard Buchner used a lysate of yeast cells to convert glucose to ethanol. Later Welch and Scopes, 1985 demonstrated cell free production of ethanol, a process which, however, was technically not useful. The system lacked specificity (side reaction of enzymes, unwanted activities in the lysate) and a maximum of 9% ethanol was obtained.
A number of technical processes have been described that use isolated enzymes for the production of chemicals. For example, alcohol dehydrogenases are used in the production of chiral alcohols from ketones. Such processes require cofactor (NAD) regeneration which can be achieved, for example, by adding glucose and glucose dehydrogenase. Such processes have been designed to produce high-value chemicals but not to provide enzyme system comprising multiple enzyme reactions that convert carbohydrates into chemicals with high energy and carbon efficiency.
Zhang et al., 2008 describe the idea for cell free enzymatic conversion of glucose to n-butanol. The concept includes a minimum of 18 enzymes, several different cofactors and coenzymes (e.g. ATP, ADP, NADH, NAD, ferredoxin and coenzyme A). In addition the postulated process results in a net-production of ATP so that it requires in addition an ATPase enzyme to remove the ATP. Under practical terms control of ATPase addition while maintaining a balanced ATP level is very difficult to achieve. In summary, the described process would be expensive, technically instable and would give only low butanol yields.
In summary there is a need for a cost effective process for the production of chemicals from renewable resources, in particular ethanol and C4 alcohols such as n-butanol and its isomers.
According to one aspect, the present invention addresses this need through a cell free enzymatic system, using only a limited number of enzymes and a limited set of cofactors. In particular, according to a preferred aspect, the inventive process does not lead to net ATP production, and/or does not use phosphorylative enzyme reactions, and/or uses only enzymes that withstand the inactivating presence of the produced chemicals.